Mitochondria are centers of metabolism and signaling whose function is essential to all but a few eukaryotic cell types. General dysfunction of these organelles is implicated in a wide range of inborn errors of metabolism, and in an increasing number of common human diseases. Elucidation of the biochemical functions of these disease-related proteins has become a bottleneck in understanding mitochondrial pathophysiology. This project aims to widen this biomedical bottleneck by accelerating the functional annotation of key disease- related orphan mitochondrial proteins (OMPs), thereby taking an important first step toward rational therapeutic design for multiple diseases. We will do so by establishing a robust computation- and mass spectrometry-based platform for systematically annotating ~100 of these disease-related OMPs, and then to employ rigorous molecular and structural biology methods to define the functions of select proteins at biochemical depth. Our annotation pipeline involves: 1) establishing an extensive network of condition-specific protein-protein interactions using affinity purification mass spectrometry in mammalian cell lines, 2) connecting the yeast orthologs of these OMPs with proteins and pathways of known function through whole-proteome and metabolome correlation profiling, and 3) interpreting these data in the context of other large-scale resources using a Bayesian framework. We will first leverage these tools to further elucidate the functions of OMPs associated with the biosynthesis of coenzyme Q (CoQ) and the assembly of complex I (CI)-core components of the mitochondrial oxidative phosphorylation machinery whose deficiency is associated with multiple human diseases. Prior studies have identified nine proteins that are associated with CoQ biosynthesis (COQ1-9), and seven associated with CI maturation/assembly (NDUFAF1-7). However, many of these proteins are OMPs with no clear biochemical roles in these pathways, and at least three steps in the CoQ pathway are likely fulfilled by yet-to-be-identified mitochondrial proteins. Our initial approaches above already have shed light on the functions of two disease-related CoQ-related OMPs (COQ8 and COQ9) and on three other novel CoQ- and CI- related proteins identified by our systematic analyses. Here, we will employ biochemical and structural biology approaches to investigate the functions of these new proteins, and to test the hypothesis that COQ9 uses its lipid-binding capacity to present CoQ intermediates to other enzymes in the pathway. In future years, we will employ similar approaches to investigate the functions of additional OMPs related to the respiratory chain in our own laboratories; will continue working with our network of collaborators that have expertise in areas distinct to our own; and will actively provide our data to the larger mitochondrial community via an interactive website to further accelerate investigations of OMPs connected to a broad range of mitochondrial processes and human diseases. In doing so, our work promises to shed new insight into basic mitochondrial function and to widen a key bottleneck in understanding mitochondrial dysfunction and its associated pathophysiology.